In recent years, organ-on-a-chip (OOC) technology has emerged as a groundbreaking platform to simulate complex physiological processes. Concurrently, the global presence of micro and nano-plastics (MNPs) in the environment and their ingestion has raised concerns about their impact on human health, specifically organs such as the lungs, liver, kidneys, and blood vessels. There is an added concern about their ability to cross even the blood-brain barrier (BBB). While numerous papers have been published assessing various environmental toxicants with OOCs, those for MNPs are relatively small. To ascertain current trends in methodologies and catalog the types of toxicants explored, we have gathered and analyzed papers that used OOCs to assess various environmental toxicants' impacts on these organs. Various platforms assessing MNPs were analyzed and compared to those for other environmental toxicants. Our results show that few articles have been published that used OOCs to assess MNPs' toxicity to human organs. Specifically, certain organs, such as the heart and skin, have little representation in this collection. OOC-based evaluation methods for MNP's toxicity have many advantages over the current methods - in vitro tests with 2D human cell cultures and animal studies - including lower cost, faster results, and greater physiological relevance. This review summarizes the current OOC techniques for assessing environmental toxicants and laboratory methods for evaluating MNPs' toxicity to humans. A systematic comparison of these methods provides a deeper understanding of the current techniques and suggests the optimized use of OOCs for assessing MNPs' and other pollutants' toxicity.

Organ-on-a-chip (OoC) devices represent advanced in vitro models enabling to mimic the human tissue architecture function and physiology, providing a promising alternative to the traditional animal testing methods. These devices combine the microfluidics with soft materials, specifically hydrogel membranes (HMs) for mimicking the extracellular matrix (ECM) and biological barriers, such as the blood-brain barrier (BBB). Hydrogels are ideal biomaterials for OoC systems because of their tunable properties, biocompatibility, biodegradability, and microscale self-assembly. The integration of HMs with OoC devices provides an effective way to develop dynamic, biologically relevant environments for supporting living cells targeted at drug discovery, disease modeling, and personalized medicine. Recent advancements in fabrication technologies such as additive manufacturing (3D printing), photolithography, and bioprinting have additionally advanced development of such systems. This review aims to outline the role of HMs in OoC platforms, highlighting their material properties, self-assembly behavior, and also challenges associated with their fabrication. Additionally, we visualize and discuss the latest progress made in utilizing HMs for applications in tissue engineering, drug development, and biosensing, with a focus on their interface dynamics and structural self-organization. The future perspective on OoC technology has also been patterned in order to provide a broader image on integration of OoC and HMs with personalized medicine and advanced drug delivery systems.

Micro and nanoplastics (MNPs) are widespread environmental and food web contaminants that are absorbed by the intestine and distributed systemically, but the mechanisms of uptake are not well understood. In a triculture small intestinal epithelial model, we previously found that uptake of 26 nm polystyrene MNPs (PS26) occurred by both passive diffusion and active actin- and dynamin-dependent mechanisms. However, studies in a more physiologically relevant model are required to validate those results. Here, a microfluidic intestine-on-a-chip model was developed using primary human intestinal epithelial organoids from healthy and Crohn's disease donors, and used to evaluate the toxicity and mechanisms effectuating uptake of 25 nm polystyrene shell-gold core tracer MNPs (AuPS25). AuPS25 caused minimal toxicity after 24 h exposure in either healthy or Crohn's IOC models. RNAseq analysis of epithelial cells identified 9 genes dysregulated by AuPS25, including downregulation of IFI6 (interferon alpha-induced protein 6). Because IFI6 has important antiviral and immunosuppressive functions in the intestine, its downregulation suggests impairment of innate immune function, which could have important negative health consequences. Inhibitor studies revealed that AuPS25 uptake in the IOC occurred by both passive diffusion and active actin- and dynamin-dependent mechanisms, consistent with our previous findings in the triculture model.

Preclinical studies are a vital component of pharmaceutical development and improvements in the predictive value of in vitro studies are essential. Organ-on-a-chip in vitro models are a recent advancement in the pursuit of improved reproduction of in vivo tissue complexity. Here, we report the development and characterization of porcine intestinal cells from organoids on chips with microfluid dynamics and peristaltic-like strain in a microphysiological system. Intestinal epithelial cells were grown on a porous membrane as a co-culture with human intestinal microvascular endothelial cells for up to 12 days. These cultures formed villi-like structures and established a tight barrier replete with F-actin and tight junctions. A demarcated region of the epithelial cells was in an actively proliferative stage, reminiscent of intestinal crypts. The intestinal epithelial cell growth was characterized for the presence of enterocytes, goblet cells and enteroendocrine cells. Notable drug transporters and CYP450 metabolic activity were present in these cultures. The organoid chip maintained barrier function as the paracellular permeability was low. In contrast, the permeability enhancer, sodium caprate (C10), increased the apparent permeability of molecular weight marker compounds by 2- to 3-fold, and upon removal of C10, the barrier was shown to be recovered. The porcine intestinal chip represents a new in vitro model with potential application in multiple aspects of pharmaceutical testing including drug metabolism, drug transporters and safety.

The biology of cancer is characterized by an intricate interplay of cells originating not only from the tumor mass, but also its surrounding environment. Different microbial species have been suggested to be enriched in tumors and the impacts of these on tumor phenotypes is subject to intensive investigation. For these efforts, model systems that accurately reflect human-microbe interactions are rapidly gaining importance. Here we present a guide for selecting a suitable in vitro co-culture platform used to model different cancer-microbiome interactions. Our discussion spans a variety of in vitro models, including 2D cultures, tumor spheroids, organoids, and organ-on-a-chip platforms, where we delineate their respective advantages, limitations, and applicability in cancer microbiome research. Particular focus is placed on methodologies that facilitate the exposure of cancer cells to microbes, such as organoid microinjections and co-culture on microfluidic devices. We highlight studies offering critical insights into possible cancer-microbe interactions and underscore the importance of in vitro models in those discoveries. We anticipate the integration of more complex microbial communities and the inclusion of immune cells into co-culture systems to more accurately simulate the tumor microenvironment. The advent of ever more sophisticated co-culture models will aid in unraveling the mechanisms of cancer-microbiome interplay and contribute to exploiting their potential in novel diagnostic and therapeutic strategies.

Caco-2 cells are derived from human colon cancer and have the ability to differentiate into human intestinal epithelial-like cells. The 2D in vitro intestinal model of Caco-2 cells cultured on a semi-permeable membrane is widely used in drug development and the evaluation of absorption functions. However, these intestinal models lack the structural characteristics of the small intestine in vivo, and the cell behavior is not properly controlled. Previous studies have reported that the microstructure of the villi and crypts on a small intestine-mimicking scaffold promotes Caco-2 differentiation; however, the effect of the nanostructure of the small intestine-mimicking scaffold on Caco-2 differentiation remains unclear. This study aimed to elucidate the effects of nanostructures on the small intestine mimetic scaffold in Caco-2 differentiation. We fabricated a decellularized small intestine in which the basement membrane nanostructure was altered through a subtractive process. Caco-2 cells were cultured on decellularized small intestine for 21 days, and the differentiation of Caco-2 cells was assessed. The microvillus density of Caco-2 cultured on decellularized small intestine that retained the unique nanostructure of small intestinal basement membrane was significantly higher than that of Caco-2 cultured on decellularized small intestine that did not retain the unique nanostructure of small intestinal basement membrane. This indicates that nanostructures specific to the basement membrane of the small intestine enhanced Caco-2 cell maturation.

Blood vessels are hierarchical microchannels that transport nutrients and oxygen to different tissues and organs, while also eliminating metabolic waste from the body. Disorders of the vascular system impact both physiological and pathological processes. Conventional animal vascular models are complex, high-cost, time-consuming, and low-validity, which have limited the exploration of effective in vitro vascular microsystems. The morphologies of micro-scaled tubular structures and physiological properties of vascular tissues, including mechanical strength, thrombogenicity, and immunogenicity, can be mimicked in vitro by engineering strategies. This review highlights the state-of-the-art and advanced engineering strategies for in vitro vascular microsystems, covering the domains related to rational designs, manufacturing approaches, supporting materials, and organ-specific cell types. A broad range of biomedical applications of in vitro vascular microsystems are also summarized, including the recent advances in engineered vascularized tissues and organs for physiological and pathological study, drug screening, and personalized medicine. Moreover, the commercialization of in vitro vascular microsystems, the feasibility and limitations of current strategies and commercially available products, as well as perspectives on future directions for exploration, are elaborated. The in vitro modeling of vascular microsystems will facilitate rapid, robust, and efficient analysis in tissue engineering and broader regenerative medicine towards the development of personalized treatment approaches.

The intestinal mucosal barrier forms a critical interface between lumen contents such as bacteria, drugs, and drug carriers and the underlying tissue. Current in vitro intestinal models, while recapitulating certain aspects of this barrier, generally present challenges with respect to imaging transport across mucus and uptake into enterocytes. A human mesofluidic small intestinal chip was designed to enable facile visualization of a mucosal interface created by growing primary human intestinal cells on a vertical hydrogel wall separating channels representing the intestinal lumen and circulatory flow. Type I collagen, fortified via cross-linking to prevent deformation and leaking during culture, was identified as a suitable gel wall material for supporting primary organoid-derived human duodenal epithelial cell attachment and monolayer formation. Addition of DAPT and PGE2 to culture medium paired with air-liquid interface culture increased the thickness of the mucus layer on epithelium grown within the device for 5 days from approximately 5 μm to 50 μm, making the model suitable for revealing intriguing features of interactions between luminal contents and the mucus barrier using live cell imaging. Time-lapse imaging of nanoparticle diffusion within mucus revealed a zone adjacent to the epithelium largely devoid of nanoparticles up to 4.5 h after introduction to the lumen channel, as well as pockets of dimly lectin-stained mucus within which particles freely diffused, and apparent clumping of particles by mucus components. Multiple particle tracking conducted on the intact mucus layer in the chip revealed significant size-dependent differences in measured diffusion coefficients. E. coli introduced to the lumen channel were freely mobile within the mucus layer and appeared to intermittently contact the epithelial surface over 30 minute periods of culture. Mucus shedding into the lumen and turnover of mucus components within cells were visualized. Taken together, this system represents a powerful tool for visualization of interactions between luminal contents and an intact live mucosal barrier.

The application of human enteroid systems presents a significant opportunity within the drug development pipeline, highlighting considerable potential for advancements in the characterization and evaluation of new molecular entities. Derived from LGR5+ crypt-based columnar cells, enteroid systems more accurately recapitulate the microanatomy and physiological processes of the human intestinal mucosa compared to traditionally used systems. They contain the complement of major mucosal epithelial cell types, maintain the genetic identity of the donor and intestinal segment they were derived from, and exhibit biological functions and specific activities that are seen in vivo. In this review, we examine the applications of human enteroid systems in nonclinical drug development and compare findings to existing and emerging in vitro models of the small intestine. Specifically, we explore enteroid systems in the context of predicting oral drug disposition, focusing on apparent permeability, intestinal first-pass metabolism, and drug interactions, as well as their utility in assessing drug-induced gastrointestinal toxicity and screening therapeutic efficacy against enteric diseases. Additionally, we highlight aspects of enteroid systems that warrant further study.

The gastrointestinal tract is a dynamic biomechanical environment where physical forces, cellular processes, and microbial interactions converge to shape the gut health and disease. In this review, we examine the unique mechanical properties of the gut, including peristalsis, viscoelasticity, shear stress, and tissue stiffness, and their roles in modulating host mechanosignaling and microbial behavior under physiological and pathological conditions. We discuss how these mechanical forces regulate gut epithelial integrity, immune responses, and microbial colonization, leading to distinct ecological niches across different intestinal segments. Furthermore, we highlight recent advancements in 3D culture systems and gut-on-a-chip models that accurately recapitulate the complex interplay between biomechanics and gut microbiota. By elucidating the intricate relationship between mechanobiology and gut function, this review underscores the potential for mechanotherapeutic strategies to modulate host-microbe interactions, offering promising avenues for the prevention and treatment of disorders such as inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer.

The advent of intestinal organoids, three-dimensional structures derived from stem cells, has significantly advanced the field of biology by providing robust in vitro models that closely mimic the architecture and functionality of the human intestine. These organoids, generated from induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or adult stem cells, possess remarkable capabilities for self-renewal, differentiation into diverse intestinal cell types, and functional recapitulation of physiological processes, including nutrient absorption, epithelial barrier integrity, and host-microbe interactions. The utility of intestinal organoids has been extensively demonstrated in disease modeling, drug screening, and personalized medicine. Notable examples include iPSC-derived organoids, which have been effectively employed to model enteric infections, and ESC-derived organoids, which have provided critical insights into fetal intestinal development. Patient-derived organoids have emerged as powerful tools for investigating personalized therapeutics and regenerative interventions for conditions such as inflammatory bowel disease (IBD), cystic fibrosis, and colorectal cancer. Preclinical studies involving transplantation of human intestinal organoids into murine models have shown promising outcomes, including functional integration, epithelial restoration, and immune system interactions. Despite these advancements, several challenges persist, particularly in achieving reproducibility, scalability, and maturation of organoids, which hinder their widespread clinical translation. Addressing these limitations requires the establishment of standardized protocols for organoid generation, culture, storage, and analysis to ensure reproducibility and comparability of findings across studies. Nevertheless, intestinal organoids hold immense promise for transforming our understanding of gastrointestinal pathophysiology, enhancing drug development pipelines, and advancing personalized medicine. By bridging the gap between preclinical research and clinical applications, these organoids represent a paradigm shift in the exploration of novel therapeutic strategies and the investigation of gut-associated diseases.

The increasing interest in utilizing three-dimensional (3D) in vitro models with innovative biomaterials to engineer functional tissues arises from the limitations of conventional cell culture methods in accurately reproducing the complex physiological conditions of living organisms. This study presents a strategy for replicating the intricate microenvironment of the intestine by cultivating intestinal cells within bioinspired 3D interfaces that recapitulate the villus-crypt architecture and 3D tissue arrangement of the intestine. Intestinal cells cultured on these biomimetic substrates exhibited phenotypes and differentiation characteristics resembling intestinal-specific cell types, effectively replicating intestinal tissue. Notably, tissue proliferation and differentiation were achieved within 72-120 h-significantly faster than the several weeks required by conventional bioengineered materials, which often pose risks of tissue necrosis or cross-contamination. Additionally, the differentiated cells on these villi-crypts mimicking bio-interfaces exhibit higher production of natural antimicrobial peptides, resulting in reduced pathogenic infection compared to control samples. Furthermore, our method stands out for simplicity in fabrication, eliminating the need for cleanroom procedures and complex microfabrication techniques.

Microfluidic devices are used for various applications in biology and medicine. From on-chip modelling of human organs for drug screening and fast and straightforward point-of-care (POC) detection of diseases to sensitive biochemical analysis, these devices can be custom-engineered using low-cost techniques. The microchannel interface is essential for these applications, as it is the interface of immobilised biomolecules that promote cell capture, attachment and proliferation, sense analytes and metabolites or provide enzymatic reaction readouts. However, common microfluidic materials do not facilitate the stable immobilisation of biomolecules required for relevant applications, making interfacial engineering necessary to attach biomolecules to the microfluidic surfaces. Interfacial engineering is performed through various immobilisation mechanisms and surface treatment techniques, which suitably modify the surface properties like chemistry and energy to obtain robust biomolecule immobilisation and long-term storage stability suitable for the final application. In this review, we provide an overview of the status of interfacial engineering in microfluidic devices, covering applications, the role of biomolecules, their immobilisation pathways and the influence of microfluidic materials. We then propose treatment techniques to optimise performance for various biological and medical applications and highlight future areas of development.

The small intestine is an area of the digestive system difficult to access using current medical procedures, which prevents studies on the interactions between food, drugs, the small intestinal epithelium, and resident microbiota. Therefore, there is a need to develop novel microfluidic models that mimic the intestinal biological and mechanical environments. These models can be used for drug discovery and disease modeling and have the potential to reduce reliance on animal models. The goal of this study was to develop a small intestine on a chip with both enterocyte (Caco-2) and goblet (HT29-MTX) cells cocultured with Lacticaseibacillus rhamnosus biofilms, which is of one of several genera present in the small intestinal microbiota. L. rhamnosus was introduced following the establishment of the epithelial barrier. The shear stress within the device was kept in the lower physiological range (0.3 mPa) to enable biofilm development over the in vitro epithelium. The epithelial barrier differentiated after 5 days of dynamic culture with cell polarity and permeability similar to the human small intestine. The presence of biofilms did not alter the barrier's permeability in dynamic conditions. Under fluid flow, the complete model remained viable and functional for more than 5 days, while the static model remained functional for only 1 day. The presence of biofilm increased the secretion of acidic and neutral mucins by the epithelial barrier. Furthermore, the small intestine on a chip also showed increased MUC2 production, which is a dominant gel-forming mucin in the small intestine. This model builds on previous publications as it establishes a stable environment that closely mimics in vivo conditions and can be used to study intestinal physiology, food-intestinal interactions, and drug development.

The high failure rates in clinical drug development based on animal models highlight the urgent need for more representative human models in biomedical research. In response to this demand, organoids and organ chips were integrated for greater physiological relevance and dynamic, controlled experimental conditions. This innovative platform-the organoids-on-a-chip technology-shows great promise in disease modeling, drug discovery, and personalized medicine, attracting interest from researchers, clinicians, regulatory authorities, and industry stakeholders. This review traces the evolution from organoids to organoids-on-a-chip, driven by the necessity for advanced biological models. We summarize the applications of organoids-on-a-chip in simulating physiological and pathological phenotypes and therapeutic evaluation of this technology. This section highlights how integrating technologies from organ chips, such as microfluidic systems, mechanical stimulation, and sensor integration, optimizes organoid cell types, spatial structure, and physiological functions, thereby expanding their biomedical applications. We conclude by addressing the current challenges in the development of organoids-on-a-chip and offering insights into the prospects. The advancement of organoids-on-a-chip is poised to enhance fidelity, standardization, and scalability. Furthermore, the integration of cutting-edge technologies and interdisciplinary collaborations will be crucial for the progression of organoids-on-a-chip technology.

Gut organoids are 3D cellular structures derived from adult or pluripotent stem cells, capable of closely replicating the physiological properties of the gut. These organoids serve as powerful tools for studying gut development and modeling the pathogenesis of intestinal diseases. This review provides an in-depth exploration of technological advancements and applications of gut organoids, with a focus on their construction methods. Additionally, the potential applications of gut organoids in disease modeling, microenvironmental simulation, and personalized medicine are summarized. This review aims to offer perspectives and directions for understanding the mechanisms of intestinal health and disease as well as for developing innovative therapeutic strategies.

Inflammatory bowel disease (IBD) is an idiopathic gastrointestinal disease with drastically increasing incidence rates. Due to its multifactorial etiology, a precise investigation of the pathogenesis is extremely difficult. Although reductionist cell culture models and more complex disease models in animals have clarified the understanding of individual disease mechanisms and contributing factors of IBD in the past, it remains challenging to bridge research and clinical practice. Conventional 2D cell culture models cannot replicate complex host-microbiota interactions and stable long-term microbial culture. Further, extrapolating data from animal models to patients remains challenging due to genetic and environmental diversity leading to differences in immune responses. Human intestine organ-on-chip (OoC) models have emerged as an alternative in vitro model approach to investigate IBD. OoC models not only recapitulate the human intestinal microenvironment more accurately than 2D cultures yet may also be advantageous for the identification of important disease-driving factors and pharmacological interventions targets due to the possibility of emulating different complexities. The predispositions and biological hallmarks of IBD focusing on host-microbiota interactions at the intestinal mucosal barrier are elucidated here. Additionally, the potential of OoCs to explore microbiota-related therapies and personalized medicine for IBD treatment is discussed.

The limitation of animal models to imitate a therapeutic response in humans is a key problem that challenges their use in fundamental research. Organ-on-a-chip (OOC) devices, also called microphysiological systems (MPS), are devices containing a lining of living cells grown under dynamic flow to recapitulate the important features of human physiology and pathophysiology with high precision. Recent advances in microfabrication and tissue engineering techniques have led to the wide adoption of OOC in next-generation experimental platforms. This review presents a comprehensive analysis of the OOC systems, categorizing them by flow types (single-pass and multipass), operational mechanisms (pumpless and pump-driven), and configurations (single-organ and multiorgan systems), along with their respective advantages and limitations. Furthermore, it explores the integration of qualitative and quantitative assay techniques, providing a comparative evaluation of systems with and without sensor integration. This review aims to fill essential knowledge gaps, driving the progress of the development of OOC systems and paving the way for breakthroughs in biomedical research, pharmaceutical innovation, and tissue engineering.

Drug development is a costly and timely process with high risks of failure during clinical trials. Although in vitro tissue models have significantly advanced over the years, thus fostering a transition from animal-derived models towards human-derived models, failure rates still remain high. Current cell-based assays are still not able to provide an accurate prediction of the clinical success or failure of a drug candidate. To overcome the limitations of current methods, a variety of microfluidic systems have been developed as powerful tools that are capable of mimicking (micro)physiological conditions more closely by integrating physiological fluid flow conditions, mechanobiological cues and concentration gradients, to name only a few. One major advantage of these biochip-based tissue cultures, however, is their ability to seamlessly connect different organ models, thereby allowing the study of organ-crosstalk and metabolic byproduct effects. This is especially important when assessing absorption, distribution, metabolism, and excretion (ADME) processes of drug candidates, where an interplay between various organs is a prerequisite. In the current review, a number of in vitro models as well as microfluidic dual- and multi-organ systems are summarized with a focus on absorption (skin, lung, gut) and metabolism (liver). Additionally, the advantage of multi-organ chips in identifying a drug's on and off-target toxicity is discussed. Finally, the potential high-throughput implementation and modular chip design of multi-organ-on-a-chip systems within the pharmaceutical industry is highlighted, outlining the necessity of reducing handling complexity.

Biological barriers formed by the endothelium and epithelium regulate nutrient exchange, disease development, and drug delivery. Organ-on-chip (OOC) systems effectively model these barriers by incorporating key biophysical cues like microscale dimensions, co-culture, and fluid flow-induced shear stress. The majority of microfluidic OOC platforms, however, require syringe and pump systems which are hindered by several limitations, including large footprints, elaborate designs, long setup times, and a high rate of failure (contamination, leakage, etc.). Here we describe VitroFlo, a pump-free microfluidic device designed for in vitro biological barrier modeling with 12 independent co-culture modules that can be simultaneously subjected to tunable, unidirectional flow with physiological shear stresses ranging from 0.01-10 dyn/cm2. We demonstrate application of the device to model vascular endothelial, blood-brain, and intestinal epithelial barriers, and confirm shear stress-dependent cell alignment, tight junction protein expression, barrier maturation, permeability, and paracrine signaling between co-cultured cells. The VitroFlo platform enables scalable and cost-effective modeling of physiological barriers to facilitate the translation of findings from in vitro systems to preclinical models.

Hematological malignancies originating from blood, bone marrow, and lymph nodes include leukemia, lymphoma, and myeloma, which necessitate the use of a distinct chemotherapeutic approach. Drug resistance frequently complicates their treatment, highlighting the need for predictive tools to guide therapeutic decisions. Conventional 2D/3D cell cultures do not fully encompass in vivo criteria, and translating disease models from mice to humans proves challenging. Organ-on-a-chip technology presents an avenue to surmount genetic disparities between species, offering precise design, concurrent manipulation of various cell types, and extrapolation of data to human physiology. The development of bone-on-a-chip (BoC) systems is crucial for accurately representing the in vivo bone microenvironment, predicting drug responses for hematological cancers, mitigating drug resistance, and facilitating personalized therapeutic interventions. BoC systems for modeling hematological cancers and drug research can encompass intricate designs and integrated platforms for analyzing drug response data to simulate disease scenarios. This review provides a comprehensive examination of BoC systems applicable to modeling hematological cancers and visualizing drug responses within the intricate context of bone. It thoroughly discusses the materials pertinent to BoC systems, suitable in vitro techniques, the predictive capabilities of BoC systems in clinical settings, and their potential for commercialization.

The intestinal microenvironment represents a complex and dynamic ecosystem, comprising a diverse range of epithelial and nonepithelial cells, a protective mucus layer, and a diverse community of gut microbiota. Understanding the intricate interplay between these components is essential for uncovering the mechanisms underlying intestinal health and disease. The development of intestinal organoids, three-dimensional (3-D) mini-intestines that closely mimic the architecture, cellular diversity, and functionality of the intestine, offers a powerful platform for investigating different aspects of intestinal physiology and pathology. However, current intestinal organoid models, mainly adult stem cell-derived organoids, lack the nonepithelial and microbial components of the intestinal microenvironment. As such, several coculture systems have been developed to coculture intestinal organoids with other intestinal elements including microbes (bacteria and viruses) and immune, stromal, and neural cells. These coculture models allow researchers to recreate the complex intestinal environment and study the intricate cross talk between different components of the intestinal ecosystem under healthy and pathological conditions. Currently, there are several approaches and methodologies to establish intestinal organoid cocultures, and each approach has its own strengths and limitations. This review discusses the existing methods for coculturing intestinal organoids with different intestinal elements, focusing on the methodological approaches, strengths and limitations, and future directions.

Non-communicable diseases (NCDs), such as type 2 diabetes (T2D) and metabolic dysfunction-associated fatty liver disease, have reached epidemic proportions worldwide. The global increase in dietary sugar consumption, which is largely attributed to the production and widespread use of cheap alternatives such as high-fructose corn syrup, is a major driving factor of NCDs. Therefore, a comprehensive understanding of sugar metabolism and its impact on host health is imperative to rise to the challenge of reducing NCDs. Notably, fructose appears to exert more pronounced deleterious effects than glucose, as hepatic fructose metabolism induces de novo lipogenesis and insulin resistance through distinct mechanisms. Furthermore, recent studies have demonstrated an intricate relationship between sugar metabolism and the small intestinal microbiota (SIM). In contrast to the beneficial role of colonic microbiota in complex carbohydrate metabolism, sugar metabolism by the SIM appears to be less beneficial to the host as it can generate toxic metabolites. These fermentation products can serve as a substrate for fatty acid synthesis, imposing negative health effects on the host. Nevertheless, due to the challenging accessibility of the small intestine, our knowledge of the SIM and its involvement in sugar metabolism remains limited. This review presents an overview of the current knowledge in this field along with implications for future research, ultimately offering potential therapeutic avenues for addressing NCDs.

Incorporating mechanical stretching of cells in tissue culture is crucial for mimicking (patho)-physiological conditions and understanding the mechanobiological responses of cells, which can have significant implications in areas like tissue engineering and regenerative medicine. Despite the growing interest, most available cell-stretching devices are not compatible with automated live-cell imaging, indispensable for characterizing alterations in the dynamics of various important cellular processes. In this work, StretchView is presented, a multi-axial cell-stretching platform compatible with automated, time-resolved live-cell imaging. Using StretchView, long-term image acquisition of cells in the relaxed and stretched states is shown for the first time (experimental time of 12 h) without the need for human intervention. Homogeneous and stable strain fields are demonstrated for 18 h of cyclic stretching, highlighting the platform's versatility and robustness. As proof-of-principle, the effect of stretching on cell kinematics and spatiotemporal localization of the cell-cell adhesion protein E-cadherin is examined for MDCK cells in monolayer. First evidence of a monotonic increase in junctional E-cadherin localization upon exposure to stretch is presented using live-cell imaging data acquired during cyclic stretching, suggestive of an increase in barrier integrity of the monolayer. These findings highlight the potential of StretchView in providing insights into cell mechanobiology and beyond.

Coeliac disease (CeD) is an immune-mediated disorder characterised by gluten-triggered inflammation and damage in the small intestine, with lifelong gluten-free diet (GFD) as the only treatment. It is a multifactorial disease, involving genetic and environmental susceptibility factors, and its complexity and lack of comprehensive human model systems have hindered understanding of its pathogenesis and development of new treatments. Therefore, it is crucial to establish systems that recapitulate patient genetic background and the interactions between the small intestinal epithelial barrier, immune cells, and environment that contribute to CeD. In this review, we discuss disease complexity, recent advances in stem cell biology, organoids, tissue co-cultures, and organ-on-chip (OoC) systems that facilitate the development of comprehensive human model systems, and model applications in preclinical studies of potential treatments.

Organoids are three-dimensional, miniaturized tissue-like structures derived from either stem cells or primary cells, emerging as powerful in vitro models for studying developmental biology, disease pathology, and drug discovery. These organoids more accurately mimic cell-cell interactions and complexities of human tissues compared to traditional cell cultures. However, challenges such as limited nutrient supply and biomechanical cue replication hinder their maturation and viability. Microfluidic technologies, with their ability to control fluid flow and mimic the mechanical environment of tissues, have been integrated with organoids to create organoid-on-chip models that address these limitations. These models not only improve the physiological relevance of organoids but also enable more precise investigation of disease mechanisms and therapeutic responses. By combining microfluidics and organoids, several advanced organoids-on-chip models have been developed to investigate mechanical and biochemical cues involved in disease progression. This review discusses various methods to develop organoids-on-chip and the recently established organoids-on-chip models with their advanced functions. Finally, we highlighted potential strategies to enhance the functionality of organoid models, aiming to overcome current limitations and bridge the gap between current cell culture models and clinical applications, advancing personalized medicine, and improving therapeutic testing.

The intestines are an important organ with a variety of functions. For drug discovery research, experimental animals and Caco-2 cells derived from a human colon carcinoma may be used to evaluate the absorption and safety of orally administered drugs. These systems have issues, such as species differences with humans in experimental animals, variations in gene expression patterns, very low drug-metabolizing activities in Caco-2 cells, and the recent trend toward reduced animal testing. Thus, there is a need for new evaluation systems. Intestinal organoid technology and microphysiological systems (MPS) have attracted attention as novel evaluation systems for predicting drug disposition, safety, and efficacy in humans in vitro. Intestinal organoids are three-dimensional structures that contain a variety of intestinal cells. They also contain crypt-villus structures similar to those of living bodies. Using MPS, it is possible to improve the functionality of cells and evaluate the linkage and crosstalk between the intestine and the liver. These systems are expected to be powerful tools for drug discovery research to predict efficacy and toxicity in humans. This review outlines the current status of intestinal organoids and MPS studies.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by non-specific inflammation. Managing UC presents significant challenges due to its chronic nature and high recurrence rates. Indigo naturalis has emerged as a potential therapeutic agent in clinical UC treatment, demonstrating advantages in alleviating refractory UC and maintaining remission periods compared to other therapeutic approaches.

This review aims to elucidate the potential mechanisms underlying the therapeutic effects of indigo naturalis in UC treatment, assess its clinical efficacy, advantages, and limitations, and provide insights into methods and strategies for utilizing indigo naturalis in UC management.

Comprehensive data on indigo naturalis were collected from reputable online databases including PubMed, GreenMedical, Web of Science, Google Scholar, China National Knowledge Infrastructure Database, and National Intellectual Property Administration.

Clinical studies have demonstrated that indigo naturalis, either alone or in combination with other drugs, yields favorable outcomes in UC treatment. Its mechanisms of action involve modulation of the AHR receptor, anti-inflammatory properties, regulation of intestinal flora, restoration of the intestinal barrier, and modulation of immunity. Despite its efficacy in managing refractory UC and prolonging remission periods, indigo naturalis treatment is associated with adverse reactions, quality variations, and inadequate pharmacokinetic investigations.

The therapeutic effects of indigo naturalis in UC treatment are closely linked to its ability to regulate the AHR receptor, exert anti-inflammatory effects, mcodulate intestinal flora, restore the intestinal barrier, and regulate immunity. Addressing the current shortcomings, including adverse reactions, quality control issues, and insufficient pharmacokinetic data, is crucial for optimizing the clinical utility of indigo naturalis in UC management. By refining patient-centered treatment strategies, indigo naturalis holds promise for broader application in UC treatment, thereby alleviating the suffering of UC patients.

Organ-on-a-chip devices are predominately made of the polymer polymethylsiloxane (PDMS), exhibiting several attractive properties e.g., transparency, gas permeability, and biocompatibility. However, the attachment of cells to this polymer has proven challenging, especially for delicate primary cells e.g., small intestinal organoid-derived epithelial cells. Hence, a need to functionalize and coat the surface has arisen to render it more hydrophilic and improve its ability to support cell adhesion and growth. While previous research has demonstrated some successful results in culturing primary cells, no comprehensive and comparative protocol has been proposed. Here, we provide a protocol for enhanced small intestinal organoid-derived epithelial cell adhesion and growth on PDMS and plastics, assessing both PDMS surface functionalization, adhesion protein coating as well as medium selection. We assess PDMS functionalization using (3-aminopropyl)trimethoxysilane (APTMS) or polyethyleneimine-glutaraldehyde (PEIGA), and adhesion protein coating using various Laminins, Collagen I, Matrigel, or mixtures thereof. Finally, we assess the use of two different medium compositions including growth factors EGF, Noggin and R-spondin1 (ENR medium) alone or combined with the two small molecules CHIR99021 and valproic acid (CV medium). We envision that our results will be useful for further attempts in emulating the small intestine using plastic- or PDMS-based devices for organs-on-a-chip development.

An organ-on-a-chip (OOAC) is a microscale device designed to mimic the functions and complexity of in vivo human physiology. Different from traditional culture systems, OOACs are capable of replicating the biochemical microenvironment, tissue-tissue interactions, and mechanical dynamics of organs thanks to the precise control offered by microfluidic technology. Diverse OOAC devices specific to different organs have been proposed for experimental research and applications such as disease modelling, personalized medicine and drug screening. Previous studies have demonstrated that the mathematical modelling of OOAC can facilitate the optimization of chips' microenvironments, serving as an essential tool to design and improve microdevices which allow reproducible growth of cell culture, reducing the time and cost of experimental testing. Here, we review recent modelling approaches for various OOAC devices, categorized according to the type of organs. We discuss the opportunities for integrating multiphysics with multicellular computational models to better characterize and predict cell culture dynamics. Additionally, we explore how developing more detailed OOAC models would support a more rapid and effective development of microdevices, and the design of robust protocols to grow and control cell cultures.

Drug-induced intestinal toxicity (GIT) is a frequent dose-limiting adverse event that can impact patient compliance and treatment outcomes. In vivo, there are proliferative and differentiated cell types critical to maintaining intestinal homeostasis. Traditional in vitro models using transformed cell lines do not capture this cellular complexity, and often fail to predict intestinal toxicity. Primary tissue-derived intestinal organoids, on the other hand, are a scalable Complex in vitro Model (CIVM) that recapitulates major intestinal cell lineages and function. Intestinal organoid toxicity assays have been shown to correlate with clinical incidence of drug-induced diarrhea, however existing studies do not consider how differentiation state of the organoids impacts assay readouts and predictivity. We employed distinct proliferative and differentiated organoid models of the small intestine to assess whether differentiation state alone can alter toxicity responses to small molecule compounds in cell viability assays. In doing so, we identified several examples of small molecules which elicit differential toxicity in proliferative and differentiated organoid models. This proof of concept highlights the need to consider which cell types are present in CIVMs, their differentiation state, and how this alters interpretation of toxicity assays.

As a model of the intestinal epithelium, intestinal stem cells (ISCs) have been grown and differentiated as monolayers on materials where stochastic organization of the crypt and villi cells occurs. We developed an allyl sulfide crosslinked photoresponsive hydrogel with a shear modulus of 1.6 kPa and functionalized with GFOGER, Bm-binder peptide ligands for monolayer growth of ISCs. The allyl sulfide chemistry allowed in situ control of mechanics in the presence of growing ISC monolayers, and structured irradiation afforded spatial regulation of the hydrogel properties. Specifically, ISC monolayers grown on 1.6 kPa substrates were in situ softened to 0.29 kPa, using circular patterns 50, 75, and 100 μm in diameter, during differentiation, resulting in control over the size and arrangement of de novo crypts and monolayer cellularity. These photoresponsive materials should prove useful in applications ranging from studying crypt evolution to drug screening and transport across tissues of changing cellular composition.

Since the development of the three-dimensional (3D) "mini-gut" culture system, adult stem cell-derived organoid technology has rapidly advanced, providing in vitro models that replicate key cellular, molecular, and physiological properties of multiple organs. The 3D intestinal organoid system has resolved many long-standing challenges associated with immortalized or cancer cell cultures, offering unparalleled capabilities for modeling gastrointestinal development and diseases. However, significant limitations remain, including restricted accessibility to the epithelial apical surface for studying host-microbe interactions, interruptions in modeling chronic gastrointestinal diseases due to frequent passaging and dissociation, and the absence of mechanical cues such as peristalsis and luminal flow, which are critical for organ development and function. To address these challenges, recent advancements have introduced Transwell-based monolayer cultures and microfluidic device-based technologies including "organ-on-a-chip" and scaffold-guided 'mini-gut' system. This review highlights these innovations, with a focus on adult stem cell-derived intestinal organoid models that feature an open apical surface and discusses their prospects and challenges for advancing basic research and clinical applications.

Representative models of intestinal diseases are transforming our knowledge of the molecular mechanisms of disease, facilitating effective drug screening and avenues for personalised medicine. Despite the emergence of 3D in vitro intestinal organoid culture systems that replicate the genetic and functional characteristics of the epithelial tissue of origin, there are still challenges in reproducing the human physiological tissue environment in a format that enables functional readouts. Here, we describe the latest platforms engineered to investigate environmental tissue impacts, host-microbe interactions and enable drug discovery. This highlights the potential to revolutionise knowledge on the impact of intestinal infection and inflammation and enable personalised disease modelling and clinical translation.

Intestinal stem cells replenish the epithelium throughout life by continuously generating intestinal epithelial cell types, including absorptive enterocytes, and secretory goblet, endocrine, and Paneth cells. This process is orchestrated by a symphony of niche factors required to maintain intestinal stem cells and to direct their proliferation and differentiation. Among the various mature intestinal epithelial cell types, Paneth cells are unique in their location in the stem cell zone, directly adjacent to intestinal stem cells. Although Paneth cells were first described as an epithelial cell component of the innate immune system due to their expression of anti-microbial peptides, they have been proposed to be niche cells due to their close proximity to intestinal stem cells and expression of niche factors. However, function as a niche cell has been debated since mice lacking Paneth cells retain functional stem cells that continue to replenish the intestinal epithelium. In this review, we summarize the intestinal stem cell niche, including the Notch, Wnt, growth factor, mechanical, and metabolic niche, and discuss how Paneth cells might contribute to these various components. We also present a nuanced view of the Paneth cell as a niche cell. Although not required, Paneth cells enhance stem cell function, particularly during intestinal development and regeneration. Furthermore, we suggest that Paneth cell loss induces intestinal stem cell remodeling to adjust their niche demands.

Mechanical stimuli have multiple effects on cell behavior, affecting a number of cellular processes including orientation, proliferation or apoptosis, migration and invasion, the production of extracellular matrix proteins, the activation and translocation of transcription factors, the expression of different genes such as those involved in inflammation and the reprogramming of cell fate. The recent development of cell stretching devices has paved the way for the study of cell reactions to stretching stimuliin-vitro, reproducing physiological situations that are experienced by cells in many tissues and related to functions such as breathing, heart beating and digestion. In this work, we review the highly-relevant contributions cell stretching devices can provide in the field of mechanobiology. We then provide the details for the in-house construction and operation of these devices, starting from the systems that we already developed and tested. We also review some examples where cell stretchers can supply meaningful insights into mechanobiology topics and we introduce new results from our exploitation of these devices.

There is an urgent need for novel methods that can accurately predict intestinal absorption of orally administered drugs in humans. This study aimed to evaluate the potential of a novel gut microphysiological system (MPS), gut MPS/Fluid3D-X, to assess the intestinal absorption of drugs in humans. The gut MPS/Fluid3D-X model was constructed using a newly developed flow-controllable and dimethylpolysiloxane-free MPS device (Fluid3D-X®). Human induced pluripotent stem cells-derived small intestinal epithelial cells were employed in this model, which exhibited key characteristics of the human absorptive epithelial cells of the small intestine, including the expression of key gene transcripts responsible for drug transport and metabolism, and the presence of dome-like protrusions in the primary intestinal epithelium under air-liquid interface culture conditions. Functional studies of transporters in the constructed model demonstrated basal-to-apical directional transport of sulfasalazine and quinidine, substrates of the active efflux transporters breast cancer resistance protein and P-glycoprotein, respectively, which were diminished by inhibitors. Furthermore, a cytochrome P450 (CYP) 3A inhibitor increased the apical-to-basal transport of midazolam, a typical CYP3A4 substrate, and reduced metabolite formation. These results suggest that gut MPS/Fluid3D-X has the potential to assess the intestinal absorption of small-molecule drugs.

Microphysiological systems (MPSs) reconstitute tissue interfaces and organ functions, presenting a promising alternative to animal models in drug development. However, traditional materials like polydimethylsiloxane (PDMS) often interfere by absorbing hydrophobic molecules, affecting drug testing accuracy. Additive manufacturing, including 3D bioprinting, offers viable solutions. GlioFlow3D, a novel microfluidic platform combining extrusion bioprinting and stereolithography (SLA) is introduced. GlioFlow3D integrates primary human cells and glioblastoma (GBM) lines in hydrogel-based microchannels mimicking vasculature, within an SLA resin framework using cost-effective materials. The study introduces a robust protocol to mitigate SLA resin cytotoxicity. Compared to PDMS, GlioFlow3D demonstrated lower small molecule absorption, which is relevant for accurate testing of small molecules like Temozolomide (TMZ). Computational modeling is used to optimize a pumpless setup simulating interstitial fluid flow dynamics in tissues. Co-culturing GBM with brain endothelial cells in GlioFlow3D showed enhanced CD133 expression and TMZ resistance near vascular interfaces, highlighting spatial drug resistance mechanisms. This PDMS-free platform promises advanced drug testing, improving preclinical research and personalized therapy by elucidating complex GBM drug resistance mechanisms influenced by the tissue microenvironment.

Alterations in intestinal structure, mechanics and physiology underlie acute and chronic intestinal conditions, many of which are influenced by dysregulation of microbiome, peristalsis, stroma or immune responses. Studying human intestinal physiology or pathophysiology is difficult in preclinical animal models because their microbiomes and immune systems differ from those of humans. Although advances in organoid culture partially overcome this challenge, intestinal organoids still lack crucial features that are necessary to study functions central to intestinal health and disease, such as digestion or fluid flow, as well as contributions from long-term effects of living microbiome, peristalsis and immune cells. Here, we review developments in organ-on-a-chip (organ chip) microfluidic culture models of the human intestine that are lined by epithelial cells and interfaced with other tissues (such as stroma or endothelium), which can experience both fluid flow and peristalsis-like motions. Organ chips offer powerful ways to model intestinal physiology and disease states for various human populations and individual patients, and can be used to gain new insight into underlying molecular and biophysical mechanisms of disease. They can also be used as preclinical tools to discover new drugs and then validate their therapeutic efficacy and safety in the same human-relevant model.

The global burden of Inflammatory bowel disease (IBD) has been rising over the last decades. IBD is an intestinal disorder with a complex and largely unknown etiology. The disease is characterized by a chronically inflamed gastrointestinal tract, with intermittent phases of exacerbation and remission. This compromised intestinal barrier can contribute to, enhance, or even enable the toxicity of drugs, food-borne chemicals and particulate matter. This review discusses whether the rising prevalence of IBD in our society warrants the consideration of IBD patients as a specific population group in toxicological safety assessment. Various in vivo, ex vivo and in vitro models are discussed that can simulate hallmarks of IBD and may be used to study the effects of prevalent intestinal inflammation on the hazards of these various toxicants. In conclusion, risk assessments based on healthy individuals may not sufficiently cover IBD patient safety and it is suggested to consider this susceptible subgroup of the population in future toxicological assessments.

Researchers have developed in vitro small intestine models of biomimicking microvilli, such as gut-on-a-chip devices. However, fabrication methods developed to date for 2D and 3D in vitro gut still have unsolved limitations. In this study, an innovative fabrication method of a 3D in vitro gut model is introduced for effective drug screening. The villus is formed on a patterned carbon nanofiber (CNF) bundle as a flexible and biocompatible scaffold. Mechanical properties of the fabricated villi structure are investigates. A microfluidic system is applied to induce the movement of CNFs villi. F-actin and Occludin staining of Caco-2 cells on a 2D flat-chip as a control and a 3D gut-chip with or without fluidic stress is observed. A permeability test of FD20 is performed. The proposed 3D gut-chip with fluidic stress achieve the highest value of Papp. Mechano-active stimuli caused by distinct structural and movement effects of CNFs villi as well as stiffness of the suggested CNFs villi not only can help accelerate cell differentiation but also can improve permeability. The proposed 3D gut-chip system further strengthens the potential of the platform to increase the accuracy of various drug tests.

Cardiovascular diseases (CVDs) are leading causes of morbidity and mortality globally, characterized by complications such as heart failure, atherosclerosis, and coronary artery disease. The vascular endothelium, forming the inner lining of blood vessels, plays a pivotal role in maintaining vascular homeostasis. The dysfunction of endothelial cells contributes significantly to the progression of CVDs, particularly through impaired cellular communication and paracrine signaling with other cell types, such as smooth muscle cells and macrophages. In recent years, co-culture systems have emerged as advanced in vitro models for investigating these interactions and mimicking the pathological environment of CVDs. This review provides an in-depth analysis of co-culture models that explore endothelial cell dysfunction and the role of cellular interactions in the development of vascular diseases. It summarizes recent advancements in multicellular co-culture models, their physiological and therapeutic relevance, and the insights they provide into the molecular mechanisms underlying CVDs. Additionally, we evaluate the advantages and limitations of these models, offering perspectives on how they can be utilized for the development of novel therapeutic strategies and drug testing in cardiovascular research.